Highly productive alpha-amylases

ABSTRACT

Provided are highly productive mutant α-amylases which are derived from an α-amylase having an amino acid sequence represented by SEQ ID No. 1 or 2 or showing at least 60% homology thereto and are constructed so that a specific amino acid residue taking part in productivity is deleted or substituted with another amino acid residue, a gene encoding the mutant α-amylase, vector, transformant cell, a method for producing the mutant α-amylase, which comprises cultivating the transformant cell, and a detergent composition containing the mutant α-amylase.  
     According to the present invention, α-amylases can be produced at a high yield from a recombinant microorganism, making it possible to drastically reduce a cost of their industrial production. This leads to production increase of liquefying alkaline α-amylases having heat resistance, chelating agent resistance and oxidant resistance and being useful as enzymes for a detergent.

DESCRIPTION

[0001] 1. Technical Field

[0002] The present invention relates to mutant α-amylases havingimproved productivity.

[0003] 2. Background Art

[0004] α-Amylases [EC.3.2.1.1.] have been used in a wide range ofindustrial fields such as starch industry, brewing industry, fiberindustry, pharmaceutical industry and food industry. Among them, thosecapable of degrading starches at high random are suited for detergents.Conventionally known as such are, as well as α-amylases derived fromBacillus licheniformis, liquefying alkaline α-amylases derived from thealkaliphilic strain Bacillus sp. KSM-AP1378 (FERM BP-3048) (WO94/26881)and improved enzymes having improved heat resistance and oxidantresistance (WO98/44126).

[0005] The present inventors have recently found liquefying alkalineα-amylases derived from the alkaliphilic strain Bacillus sp. KSM-K38(FERM BP-6946) and having chelating-agent- and oxidation-resistance(Japanese Patent Application No. Hei 10-362487, Japanese PatentApplication No. Hei 10-362488); and improved enzymes having improvedheat resistance (Japanese Patent Application No. Hei 11-163569).

[0006] In addition to such properties, enzymes for detergents arerequired to have high productivity in consideration of their industrialproduction. Although various trials have been made to improve the heatresistance or oxidant resistance of α-amylases for detergent by usingprotein engineering technique, neither improvement of productivity hasbeen considered sufficiently nor an attempt of production increase bymutation of a structural gene has been reported yet.

[0007] An object of the present invention is to provide mutantα-amylases having excellent productivity.

DISCLOSURE OF THE INVENTION

[0008] The present inventors introduced, in microorganisms, mutantα-amylase structural gene constructed by site-directed mutagenesis andevaluated productivity of α-amylases. As a result, it has been foundthat since an α-amylase gene has a site taking part in the improvementof productivity, introduction, into a microorganism, of a recombinantgene having this site mutated makes it possible to produce α-amylaseshaving drastically improved productivity.

[0009] In one aspect of the present invention, there is thus provided amutant α-amylase which is derived from an α-amylase having an amino acidsequence represented by SEQ ID No. 1 or showing at least 60% homologythereto by substitution or deletion of at least one amino acid residuecorresponding to any one of Pro₁₈, Gln₈₆, Glu₁₃₀, Asnl₁₅₄, Arg₁₇₁,Ala₁₈₆, Glu₂₁₂, Val₂₂₂, Tyr₂₄₃, Pro₂₆₀, Lys₂₆₉, GlU₂₇₆, Asn₂₇₇, Arg₃₁₀,Glu₃₆₀, Gln₃₉₁, Trp₄₃₉, Lys₄₄₄, Asn₄₇₁ and Gly₄₇₆ of the amino acidsequence.

[0010] In another aspect of the present invention, there is alsoprovided a mutant α-amylase derived from an α-amylase having an aminoacid sequence represented by SEQ ID No. 2 or showing at least 60%homology thereto by substitution or deletion of at least one amino acidresidue corresponding to any one of Asp₁₂₈, Gly₁₄₀, Ser₁₄₄, Arg₁₆₈,Asn₁₈₁, Glu₂₀₇, Phe₂₇₂, Ser₃₇₅, Trp₄₃₄ and Glu₄₆₆ of the amino acidsequence.

[0011] In a further aspect of the present invention, there is alsoprovided a gene encoding this mutant α-amylase, a vector containing thegene, a cell transformed with the vector and a production method of amutant α-amylase which comprises cultivating the transformed cell.

[0012] In a still further aspect of the present invention, there is alsoprovided a detergent composition containing this mutant α-amylase.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 illustrates a method of constructing a recombinant plasmidfor production of an α-amylase derived from the strain KSM-1378 orKSM-K38.

[0014]FIG. 2 is a schematic view illustrating a method of introducing amutation into an α-amylase gene derived from the strain KSM-1378 orKSM-K38.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The term “highly productive mutant α-amylase” as used hereinmeans an α-amylase whose yield is increased, upon production of it bycultivating a recombinant microorganism, by at least 5%, preferably atleast 10%, more preferably at least 20% compared with that beforemutation.

[0016] The mutant α-amylase of the present invention is constructed sothat out of amino acids constituting the α-amylase, the amino acidresidues taking part in the productivity are substituted with anotheramino acid residues or deleted. Examples of the α-amylase usable hereinclude liquefying α-amylases derived from Bacillus. amyloliquefaciensor Bacillus. licheniformis and liquefying alkaline α-amylases derivedfrom alkaliphilic microorganisms belonging to the Bacillus sp., of whichα-amylases having an amino acid sequence represented by SEQ ID No. 1 orSEQ ID No. 2 and α-amylases having at least 60% homology to theabove-described amino acid sequence are preferred.

[0017] Examples of the α-amylase having the amino acid sequencerepresented by SEQ ID No. 1 or α-amylase having at least 60% homologythereto include liquefying alkaline α-amylases derived from the strainBacillus sp. KSM-AP1378 (FERM BP-3048) (Japanese Patent ApplicationLaid-Open No. Hei 8-336392) and improved enzymes of the above-describedone in heat resistance and oxidant resistance which are constructed byprotein engineering technique (WO98/44126).

[0018] Examples of the α-amylase having the amino acid sequencerepresented by SEQ ID No. 2 or having at least 60% homology theretoinclude liquefying alkaline α-amylases derived from the strain Bacillussp. KSM-K38 (FERM BP-6946) and improved enzymes of the above-describedone in heat resistance which are constructed by protein engineeringtechnique (Japanese Patent Application No. Hei 11-163569).

[0019] The homology of an amino acid sequence is calculated byLipman-Pearson method (Science, 227, 1435(1985)).

[0020] The mutant α-amylase of the present invention can be obtainedfirst by cloning, from a microorganism producing an α-amylase, a geneencoding the α-amylase. For this purpose, ordinarily employed generecombinant technique, for example, the method as described in JapanesePatent Application Laid-Open No. Hei 8-336392 may be employed. Examplesof the gene usable here include that represented by SEQ ID No. 3 or SEQID No. 4 which encodes the amino acid sequence represented by SEQ ID No.1 or SEQ ID No. 2. Mutant genes derived from the above-described onesand having improved heat resistance and oxidant resistance are alsousable.

[0021] For mutation of the gene thus obtained by cloning, anysite-directed mutagenesis ordinarily employed can be adopted. Forexample, mutation can be conducted using a “Site-Directed MutagenesisSystem Mutan-Super Express Km” kit (product of Takara Shuzo Co., Ltd.).

[0022] Mutation for obtaining highly productive α-amylases of theinvention can be attained, for example, by substitution or deletion, inan α-amylase having an amino acid sequence represented by SEQ ID No. 1or having at least 60% homology thereto, of at least one amino acidresidue corresponding to any one of Pro₁₈, Gln₈₆, Glu₁₃₀, Asn₁₅₄,Arg₁₇₁, Ala₁₈₆, Glu₂₁₂, Val₂₂₂, Tyr₂₄₃, Pro₂₆₀, Lys₂₆₉, Glu₂₇₆, Asn₂₇₇,Arg₃₁₀, Glu₃₆₀, Gln₃₉₁, Trp₄₃₉, Lys₄₄₄, Asn₄₇₁ and Gly₄₇₆ of the aminoacid sequence; or by substitution or deletion, in another α-amylasehaving an amino acid sequence represented by SEQ ID No. 2 or having atleast 60% homology thereto, of at least one amino acid residuecorresponding to any one of AsP₁₂₈, Gly₁₄₀, Ser₁₄₄, Arg₁₆₈, Asn₁₈₁,Glu₂₀₇, Phe₂₇₂, Ser₃₇₅, Trp₄₃₄ and Glu₄₆₆ of the amino acid sequence.Preferred mutations include, in the amino acid sequence of SEQ ID No. 1,substitution of the amino acid residue corresponding to Pro₁₈ with Seror Thr, the amino acid residue corresponding to Gln₈₆ with Glu, theamino acid residue corresponding to Glu₁₃₀ with Val or Gln, the aminoacid residue corresponding to Asn₁₅₄ with Asp, the amino acid residuecorresponding to Arg₁₇₁ with Cys or Gln, the amino acid residuecorresponding to Ala₁₈₆ with Val or Asn, the amino acid residuecorresponding to Glu₂₁₂ with Asp, the amino acid residue correspondingto Val₂₂₂ with Glu, the amino acid residue corresponding to Tyr₂₄₃ withCys or Ser, the amino acid residue corresponding to Pro₂₆₀ with Glu, theamino acid residue corresponding to Lys₂₆₉ with Gln, the amino acidresidue corresponding to Glu₂₇₆ with His, the amino acid residuecorresponding to Asn₂₇₇ with Ser or Phe, the amino acid residuecorresponding to Arg₃₁₀ with Ala, the amino acid residue correspondingto Glu₃₆₀ with Gln, the amino acid residue corresponding to Gln₃₉₁ withGlu, the amino acid residue corresponding to Trp₄₃₉ with Arg, the aminoacid residue corresponding to Lys₄₄₄ with Arg, the amino acid residuecorresponding to Asn₄₇₁ with Asp or Glu, or the amino acid residuecorresponding to Gly₄₇₆ with Asp;

[0023] or substitution, in the amino acid sequence of SEQ ID No. 2, ofthe amino acid residue corresponding to Asp₁₂₈ with Val or Gln, theamino acid residue corresponding to Gly₁₄₀ with Ser, the amino acidresidue corresponding to Ser₁₄₄ with Pro, the amino acid residuecorresponding to Arg168 with Gln, the amino acid residue correspondingto Gln₁₈₁with Val, the amino acid residue corresponding to Glu₂₇₀ withAsp, the amino acid residue corresponding to Phe₂₇₂ with Ser, the aminoacid residue corresponding to Ser₃₇₅ with Pro, the amino acid residuecorresponding to Trp₄₃₄ with Arg or the amino acid residue correspondingto Glu₄₆₆ with Asp.

[0024] Among the mutations of the amino acid sequence of SEQ ID No. 1,those by substitution of the amino acid residue corresponding to Gln₈₆with Glu, the amino acid residue corresponding to Glu₁₃₀ with Val orGln, the amino acid residue corresponding to Ala₁₈₆ with Asn, the aminoacid residue corresponding to Tyr₂₄₃ with Ser, the amino acid residuecorresponding to Pro₂₆₀ with Glu, the amino acid residue correspondingto Lys₂₆₉ with Gln, the amino acid residue corresponding to Asn₂₇₇ withPhe and the amino acid residue corresponding to Asn₄₇₁ with Asp or Glucan bring about improvement in solubility of the α-amylase in a culturemedium or desalted and concentrated solution thereof. More specifically,the above-described mutations make it possible to improve the residualactivity of the α-amylase in the supernatant after storage at 4° C. forone week in a desalted and concentrated solution by at least 5%,especially 10% compared with the activity before mutation. Accordingly,in the case of the mutant α-amylases of the present invention obtainedby such amino acid mutations, a fermented concentrate solution of a highconcentration is available at a high yield and enzyme formulationtreatment such as ultrafiltration after fermentation production can beconducted efficiently.

[0025] A combination of two or more substitutions or deletions ofvarious amino acid residues is also effective for such amino acidmutations. It is also possible to use the above-exemplified mutation incombination with a mutation for improving enzymatic properties, forexample, in an α-amylase having an amino acid sequence represented bySEQ ID No. 1 or having at least 60% homology thereto, a mutation forimproving heat resistance by deleting amino acid residues correspondingto Arg₁₈₁ and Gly₁₈₂, a mutation for improving oxidant resistance bysubstituting the amino acid residue corresponding to Met₂₂₂ with Thr ora mutation for improving solubility by substituting the amino acidresidue corresponding Lys₄₈₄ with Gln; or in an α-amylase having anamino acid sequence represented by SEQ ID No. 2 or having at least 60%homology thereto, a mutation for further reinforcing oxidant resistanceby substituting the amino acid residue corresponding to Met₁₀₇ with Leuor a mutation for heightening detergency of a laundry detergent bysubstituting the amino acid residue corresponding Glu₁₈₈ with Ile.

[0026] A mutant α-amylase is available at a high yield by appropriatelycombining a mutant α-amylase structural gene with a control gene and aproper plasmid vector, thereby constructing a plasmid for the productionof the α-amylase, introducing the resulting plasmid into a microorganismsuch as Bacillus subtilis or Escherichia coli, preferably, Bacillussubtilis and cultivating the resulting recombinant microorganism.

[0027] The mutant α-amylase thus obtained has improved productivity byabout 10 to 500% as shown later in Examples while maintainingbiochemical properties as an enzyme, thus having excellent properties.By the above-described mutation of the amino acid residues of liquefyingalkaline α-amylases having heat resistance, chelating agent resistance,oxidant resistance and high solubility, it is therefore possible tocreate useful enzymes having drastically improved productivity in arecombinant microorganism without losing the above-described originalproperties.

[0028] The detergent compositions of the present invention may contain,in addition to the α-amylase of the invention, one or more than oneenzymes selected from debranching enzymes (such as pullulanase,isoamylase and neopullulanase), α-glucosidase, glucoamylase, protease,cellulase, lypase, pectinase, protopectinase, pectate lyase, peroxidase,laccase and catalase.

[0029] The detergent composition may contain, in addition, componentsordinarily added to a detergent, for example, surfactants such asanionic surfactants, amphoteric surfactants, nonionic surfactants andcationic surfactants, chelating agents, alkali agents, inorganic salts,anti-redeposition agents, chlorine scavengers, reducing agents,bleaching agents, fluorescent dye solubilizing agents, perfumes,anti-caking agents, enzyme activating agents, antioxidants, antiseptics,blueing agents, bleach activating agents, enzyme stabilizing agents andregulator.

[0030] The detergent compositions of the invention can be produced in amanner known per se in the art from a combination of the highlyproductive α-amylase available by the above-described method and theabove-described known detergent components. The form of the detergentcan be selected according to the using purpose and examples includeliquid, powder and granule. The detergent compositions of the presentinvention are suited as laundry detergents, bleaching detergents,detergents for automatic dish washer, pipe cleaners, and artificialtooth cleaners, of which they are especially suited as laundrydetergents, bleaching detergents and detergents for automatic dishwasher.

[0031] The highly productive mutant α-amylases of the invention are alsousable as starch liquefying saccharifying compositions. Moreover, thesemutant α-amylases, after addition thereto of one or more than oneenzymes selected from glucoamylase, maltase, pullulanase, isoamylase andneopullulanase, can be allowed to act on starches.

[0032] Furthermore, the mutant α-amylases of the present invention areusable as a desizing composition of fibers and an enzyme such aspullulanase, isoamylase or neopullulanase can be incorporated in thecomposition.

EXAMPLES

[0033] Measurement of Amylase Activity and Protein Content

[0034] Amylase activity and protein content of the enzymes each producedfrom recombinant Bacillus subtilis were measured in accordance with thebelow-described methods.

[0035] Amylase activity was measured by the 3,5-dinitrosalicylic acidmethod (DNS method). After reaction at 50° C. for 15 minutes in areaction mixture of a 40 mM glycine-sodium hydroxide buffer (pH 10)containing soluble starch, the reducing sugar thus formed wasquantitatively analyzed by the DNS method. As the titer of the enzyme,the amount of the enzyme which formed reducing sugar equivalent to 1μmol of glucose in one minute was defined as one unit.

[0036] The protein content was determined by “Protein Assay Kit”(product of Bio-Rad Laboratories) using bovine serum albumin asstandard.

Referential Example 1

[0037] Screening of Liquefying Alkaline Amylase

[0038] About 0.5 g of soil was suspended in sterilized water and theresulting suspension was heat treated at 80° C. for 15 minutes. Thesupernatant of the heat treated mixture was diluted with an adequateamount of sterilized water, followed by applying to an isolating agarmedium (Medium A). The medium was then cultured at 30° C. for 2 days togrow colonies. The colonies which formed transparent zones in theirperipheries due to starch dissolution were selected and isolated asamylase producing strains. The resulting isolated strains wereinoculated in Medium B, followed by aerobic shaken culture at 30° C. for2 days. After cultivation, the chelating agent (EDTA) resisting capacityof the supernatant obtained by centrifugation was measured and inaddition, the optimum working pH was measured. Thus, strain Bacillus sp.KSM-K38 (FERM BP-6946) was obtained. Medium A: Tryptone 1.5% Soytone0.5% Sodium chloride 0.5% Colored starch 0.5% Agar 1.5% Na₂Co₃ 0.5% (pH10) Medium B: Tryptone 1.5% Soytone 0.5% Sodium chloride 0.5% Solublestarch 1.0% Na₂CO₃ 0.5% (pH 10)

[0039] The mycological properties of strain KSM-K38 are shown inTable 1. TABLE 1 Strain KSM-K38 (a) Observation under microscope Cellsare rods of a size of 1.0 to 1.2 μm × 2.4 to 5.4 μm in the strain K36and 1.0 to 1.2 μm × 1.8 to 3.8 μm in the strain K38, and form anelliptical endospore (1.0 to 1.2 μm × 1.2 to 1.4 μm) at theirsubterminals or center. They have flagella and are motile. Gram'sstaining is positive. Acid fastness: negative. (b) Growth in variousculture mediums. The strains are alikaliphilic so that 0.5% sodiumcarbonate was added to the culture medium in the tests describedhereinafter. Nutrient agar plate culture Growth of cells is good. Colonyhas a circular shape, with its surface being smooth and its peripheralend being smooth. The color of the colony is yellowish brown. Nutrientagar slant culture Cells can grow. Nutrient broth Cells can grow. Stabculture in nutrient-broth gelatin Growth of cells is good. Liquefactionof gelatin is not observed. Litmus milk medium No change in growth. (c)Physiological properties Nitrate reduction and denitrification Nitratereduction: positive Denitrification: negative MR test Indeterminablebecause the medium is an alkaline medium. V-P test Negative Productionof indole Negative Production of hydrogen sulfide Negative Hydrolysis ofstarch Positive Utilization of citric acid Positive in Christensen'smedium but negative in Koser's medium and Simmon's medium. Utilizationof inorganic nitrogen sources Nitrate is utilized but ammonium salts arenot. Production of colorants Negative Urease Negative Oxidase NegativeCatalase Positive Growth range Growth temperature range: 15 to 40° C.,optimum growth temperature: 30° C., growth pH range: pH 9.0 to 11.0,optimum growth pH range: same Behavior on oxygen Aerobic O-F test Cellsdo not grow Sugar utilization L-galactose, D-xylose, L-arabinose,lactose, glycerin, melibiose, ribose, D-glucose, D-mannose, maltose,sucrose, trehalose, D-mannitol, starch, raffinose and D-fructose areutilized. Growth in a salt-containing medium Cells can grow when saltconcentration is 12%, but not when salt concentration is 15%.

Referential Example 2

[0040] Cultivation of Strain KSM-K38

[0041] In the liquid medium B of Referential Example 1, the strainKSM-K38 was inoculated, followed by aerobic shaken culture at 30° C. for2 days. The amylase activity (at pH 8.5) of each of the supernatantsisolated by centrifugation was measured. As a result, the activity in 1L of the culture medium was found to be 1177 U.

Referential Example 3

[0042] Purification of Liquefying Alkaline Amylase

[0043] Ammonium sulfate was added to the supernatant of the culturemedium of the strain KSM-K38 obtained in Referential Example 2 to give80% saturation, followed by stirring. The precipitate thus formed wascollected and dissolved in a 10 mM tris-HCl buffer (pH 7.5) containing 2mM CaCl₂ to dialyze the resulting solution against the buffer overnight.The dialysate was loaded on a DEAE-Toyopearl 650M column equilibratedwith the same buffer and protein was eluted in a linear gradient of 0 to1 M of NaCl in the same buffer. The active fraction obtained by gelfiltration column chromatography after dialysis against the same bufferwas dialyzed against the buffer, whereby purified enzyme exhibited asingle band on polyacrylamide gel electrophoresis (gel concentration:10%) and sodium dodecylsulfate (SDS) electrophoresis was obtained.

Referential Example 4

[0044] Enzymological Properties

[0045] The properties of the purified enzyme are as follows:

[0046] (1) Action

[0047] It acts on starch, amylose, amylopectin and α-1,4-glycoside bondwhich is a partially degraded product thereof to degrade them andproduce, from amylose, glucose (G1), maltose (G2), maltotriose (G3),maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) andmaltoheptaose (G7). But it does not act on pullulan.

[0048] (2) pH Stability (Britton-Robinson Buffer)

[0049] It exhibits residual activity of 70% or more within a range of pH6.5 to 11.0 under treating conditions at 40° C. for 30 minutes.

[0050] (3) Working Temperature Range and Optimum Working Temperature

[0051] It acts in a wide temperature range of from 20 to 80° C., withthe optimum working temperature being 50 to 60° C.

[0052] (4) Temperature Stability

[0053] The temperature at which the enzyme loses its activity wasexamined by causing a temperature change in a 50 mM glycine-sodiumhydroxide buffer (pH 10) and then, treating at each temperature for 30minutes. The residual activity of the enzyme is 80% or more at 40° C.and about 60% even at 45° C.

[0054] (5) Molecular Weight

[0055] The molecular weight as measured by sodium-dodecylsulfatepolyacrylamide gel electrophoresis is 55,000 ±5,000.

[0056] (6) Isoelectric Point

[0057] Its isoelectric point as measured by isoelectric focusingelectrophoresis is about 4.2.

[0058] (7) Effects of Surfactants

[0059] It is almost free from activity inhibition (activity remainingratio: 90% or more) even when treated at pH 10 and 30° C. for 30 minutesin a 0.1% solution of a surfactant such as sodium linear alkylbenzenesulfonate, alkyl sulfate ester sodium salt, polyoxyethylene alkylsulfateester sodium salt, sodium α-olefin sulfonate, sodium α-sulfonated fattyacid ester, sodium alkylsulfonate, SDS, soap and softanol.

[0060] (8) Effects of Metal Salts

[0061] It was treated at pH 10 and 30° C. for 30 minutes in each of thereaction systems containing various metal salts and their effects werestudied. Its activity is inhibited by 1 mM of Mn²⁺ (inhibition ratio:about 75%) and slightly inhibited by 1 mM of Sr²⁺ and Cd²⁺ (inhibitionratio: about 30%).

Example 1

[0062] Preparation of Various Recombinant Plasmids Having an α-AmylaseGene Ligated Thereto

[0063] In accordance with the method as described in WO98/44126, genesencoding a mutant α-amylase (which will hereinafter be described as“ΔRG”) having improved heat resistance and a mutant α-amylase(“ΔRG-M202T”) having improved oxidant resistance as well as improvedheat resistance were constructed, respectively, by deleting Arg₁₈₁ andGly₁₈₂ of the α-amylase (“LAMY”) which was derived from the strainBacillus sp. KSM-AP1378 (FERM BP-3048) and had the amino acid sequencerepresented by SEQ ID No. 1; and by, in addition to this mutation bydeletion, substituting Thr for Met₂₀₂ of the amino acid sequencerepresented by SEQ ID No. 1. With the genes as a template, genefragments (about 1.5 kb) encoding these mutant α-amylases were amplifiedby the PCR reaction using primers LAUS (SEQ ID No. 5) and LADH (SEQ IDNo. 6). After cutting of them with a restriction enzyme SalI, each ofthe fragments was inserted into the SalI-SmaI site of an expressionvector pHSP64 (Japanese Patent Application Laid-Open No. Hei 6-217781),whereby a recombinant plasmid having a structural gene of each of themutant α-amylases bonded thereto was constructed downstream of a strongpromoter derived from an alkaline cellulase gene of the strain Bacillussp. KSM-64 (FERM P-10482).

[0064] In the meantime, with a chromosomal DNA, which had been extractedfrom the cells of the strain Bacillus sp. KSM-K38 (FERM BP-6946) by themethod of Saito and Miura (Biochim. Biophys. Acta, 72, 619(1961)), as atemplate, PCR reaction was effected using primers K38US (SEQ ID No. 7)and K38DH (SEQ ID No. 8) shown in Table 2, whereby a structural genefragment (about 1.5kb) encoding an α-amylase (which will hereinafter bedescribed as “K38AMY”) having an amino acid sequence of SEQ ID No. 2 wasamplified. After cutting of it with a restriction enzyme SalI, theresulting fragment was inserted into the SalI-SmaI site of an expressionvector pHSP64 to construct, downstream of a strong promoter derived froman alkaline cellulase gene of the strain Bacillus sp. KSM-64 (FERMP-10482) contained in pHSP64, a recombinant plasmid having a structuralgene of K38AMY bonded thereto (FIG. 1). With this recombinant plasmid asa template, PCR reaction was effected using the primers CLUBG (SEQ ID.No. 9) and K38DH (SEQ. ID. 8) to amplify a gene fragment (about 2.1 kb)having the strong promoter and K38AMY bonded thereto.

[0065] By the recombinant PCR method as described below, a gene encodingchimeric α-amylase between K38AMY and LAMY was constructed. Describedspecifically, with a chromosomal DNA of the strain KSM-K38 (FERM BP6946)as a template, PCR reaction was conducted using primers K38DH (SEQ IDNo. 8) and LA-K38 (SEQ ID No. 10) shown in Table 2, whereby a fragmentencoding the sequence from Gln₂₀ downstream to the C-terminal of theamino acid sequence of K38AMY represented by SEQ ID No. 2 was amplified.With the above-described recombinant plasmid containing the LAMY geneand strong promoter as a template, PCR reaction was conducted usingprimers CLUBG (SEQ ID No. 9) and LA-K38R (SEQ ID No. 11) shown in Table2, whereby a gene fragment encoding from the upstream strong promoter toGly₂₁ of the amino acid sequence of LAMY of SEQ ID No. 1 was amplified.By the second PCR reaction using the resulting two DNA fragments andprimers CLUBG (SEQ ID No. 9) and K38DH (SEQ ID No. 8) shown in Table 2,the resulting two fragments having, at the end thereof, complementarysequences derived from primers LA-K38 (SEQ ID No. 10) and LA-K38R (SEQID No. 11) respectively were combined, whereby a gene fragment (about2.1 kb) encoding a chimeric α-amylase (which will hereinafter bedescribed as “LA-K38AMY”) which has, successively bonded thereto, aregion encoding from His₁ to Gly₂₁ of the LAMY downstream of the strongpromoter and a region encoding from Gln₂₀ to the C-terminal of theK38AMY was amplified.

[0066] By using a “Site-Directed Mutagenesis System Mutan-Super ExpressKm” kit (product of Takara Shuzo Co., Ltd.), the below-describedmutations were introduced to the K38AMY and LA-K38AMY. First, the K38AMYand LA-K38AMY gene fragments (about 2.1 kb) were inserted into the siteSmaI of a plasmid vector pKF19k attached to the kit to construct amutagenic recombinant plasmid (FIG. 2). A site-directed mutagenicoligonucleotide primer N190F (SEQ ID No. 50) shown in Table 2 was5′-phosphorylated with T4 DNA kinase. Using this and the above-describedmutagenic recombinant plasmid, mutagenesis was effected in accordancewith the method of the kit and by using the reaction product, the strainEscherichia coli MV1184 (“Competent cell MV1184”, product of TakaraShuzo Co., Ltd.) was transformed. From the transformant thus obtained, arecombinant plasmid was extracted, followed by analysis of a basicsequence, whereby mutation by substitution of Phe for Asn₁₉₀ wasconfirmed. By repeated introduction of mutagenic reactions into themutated gene by successively using primers A209V (SEQ ID No. 51) andQEYK (SEQ ID No. 49) in a similar manner as above, thereby substitutingAsn₁₉₀ and Gln₂₀₉, each of the amino acid sequence of the K38AMYrepresented by SEQ ID No. 2, with Phe and Val, respectively, and thesequence from Asp₁ to Gly₁₉ of the amino acid sequence of the K38AMYrepresented by SEQ ID No. 2 with the sequence from His₁ to Gly₂₁ of theamino acid sequence of the LAMY represented by SEQ ID NO. 1; bysubstituting Gln₁₆₇, Tyr₁₆₉, Asn₁₉₀ and Gln₂₀₉, each of the amino acidsequence of the K38AMY, with Glu, Lys, Phe and Val, respectively and thesequence from Asp1 to Gly₁₉ of the amino acid sequence of the K38AMYwith the sequence from His₁ to Gly₂₁ of the amino acid sequence of theLAMY; and substituting Gln₁₆₇ and Tyr₁₆₉, Asn₁₉₀ and Gln₂₀₉, each of theamino acid sequence of the K38AMY, with Glu, Lys, Phe and Val,respectively, genes encoding a mutant α-amylase (which will hereinafterbe described as “LA-K38AMY/NFQV”) having improved heat resistance, amutant α-amylase (“LA-K38AMY/QEYK/NFQV”) having drastically improvedheat resistance, and a mutant α-amylase (“QEYK/NFQV”) having improvedheat resistance were constructed, respectively.

[0067] With these genes as a template, PCR reaction was conducted usingprimers K38US (SEQ ID No. 7) and K38DH (SEQ ID No. 8) to amplifystructural gene fragments (about 1.5 kb) encoding the mutant α-amylaseswere amplified. They were then inserted into the SalI-SmaI site of anexpression vector pHSP64 in a similar manner as above, whereby arecombinant plasmid having structural genes of these mutant α-amylasesbonded each other was constructed (FIG. 1).

Example 2

[0068] Introduction of a Mutation for Improving α-Amylase Productivity

[0069] A “Site-Directed Mutagenesis System Mutan-Super Express Km” kitof Takara Shuzo Co., Ltd. was used for site-directed mutagenesis forimproving amylase productivity of recombinant Bacillus subtilis. Withvarious recombinant plasmids obtained in Example 1 as a template, PCRreactions were effected using primers CLUBG (SEQ ID No. 9) and LADH (SEQID No. 6) for ARG and ΔRG/M202T, while using primers CLUBG (SEQ ID No.9) and K38DH (SEQ ID No. 8) for K38AMY, LA-K38AMY/NFQV,LA-K38AMY/QEYK/NFQV and QEYK/NFQV, whereby fragments of about 2.1 kbfrom the upstream strong promoter derived from the strain KSM-64 to thedownstream α-amylase gene were amplified. These amplified fragments wereinserted into the SmaI site of a plasmid vector pKF19k attached to theabove-described kit, whereby various mutagenetic recombinant plasmidswere constructed (FIG. 2).

[0070] Various oligonucleotide primers for site-directed mutagenesisshown in Table 2 (SEQ ID Nos. 12 to 51) were 5′-phosphorylated withT4DNA kinase, and by using the resultant products and the abovemutagenetic recombinant plasmids, mutagenesis was conducted inaccordance with the method as described in the kit. With the reactionproducts, the strain Escherichia coli MV1184(“Competent Cell MV1184”product of Takara Shuzo Co., Ltd.) was transformed. From the resultingtransformants, a recombinant plasmid was extracted, followed by analysisof a base sequence to confirm mutation. TABLE 2 SeQ ID Using No. PrimerBase sequence (5′-3′) purpose 5 LAUS GAGTCGACCAGCACAAGCCCATCATAATGG PCRfor 6 LADH TAAAGCTTCAATTTATATTGG recombi- 7 K38USGGGTCGACCAGCACAAGCCGATGGATTGAACGGTACGATG nation 8 K38DHTAAAGCTTTTGTTATTGGTTCACGTACAC 9 CLUBG CCAGATCTACTTACCATTTTAGAGTCA 10LA-K38 ATTTGCCAAATGACGGGCAGCATTGGAATCGGTT 11 LA-K38RAACCGATTCCAATGCTGCCCGTCATTTGGCAAAT 12 P18STTTGAATGGCATTTGTCAAATGACGGGGAACCAC Site-directed 13 Q86EACAAGGAGTCAGTTGGAAGGTGCCGTGACATCT mutagenesis 14 E130VCGAAACCAAGTAATATCAGGT (ΔRG) 15 N154D AATACCCATTCCGATTTTAAATGGCGC 16R171C GATTGGGATCAGTCATGYCAGCTTCAGAACAAA 17 A186VAAATTCACCGGAAAGGTATGGGACTGGGAAGTA 18 E212D TCATCCAGATGTAATCAATG 19 V222ECTTAGAAATTGGGGAGAATGGTATACAAATACA 20 Y243CGTGAAACATATTAAATGCAGCTATACGAGAGAT 21 P260EAACACCACAGGTAAAGAAATGTTTGCAGTTGCA 22 K269Q AGAATTTTGGCAAAATGACCT 23E276H TTGCTGCAATCCATAACTATTTAAAT 24 N277SCTTGCTGCAATCGAAAGYTATTTAAATAAAACA 25 R310AGGCTATTTTGATATGGCAAATATTTTAAATGGT 26 E360Q TCTGACAAGGCAGCAAGGTTA 27Q391E GATCCACTTCTGGAAGCACGTCAAACG 28 W439R GGGGGTAATAAAAGAATGTATGTCGGG29 K444R ATGTATGTCGGGCGACATAAAGCTGG 30 N471D GATGGTTGGGGGGATTTCACTGTAA31 G476D TTCACTGTAAACGATGGGGCAGTTTCG 32 K484Q GGTTTGGGTGCAGCAATAAAT 33P18X TTTGAATGGCATTTGNNNAATGACGGGAACCAC Site-directed 34 A186XAAATTCACCGGAAAGNNNTGGGACTGGGAAGTA mutagenesis 35 Y243XGTGAAACATATTAAANNNAGCTATACGAGAGAT (for ΔRG/ 36 N277XCTTGCTGCAATCGAANNNTATTTAAATAAAACA M2027) 37 N471EGATGGTTGGGGGGAATTCACTGTAA 38 D128VCCAACGAATCGTTGGCAGGTAATTTCAGGTGCCTACACG Site-directed 39 G140SATTGATGCGTGGACGAGTTTCGACTTTTCAGGG mutagenesis 40 S144PTTTCGACTTTCCAGGGCGTAA for 41 R168QGGTGTTGACTGGGATCAGCAATATCAAGAAAATCATATTTTCC K38AMY) 42 N181VCATATTTTCCGCTTTGCAAATACGGTNTGGAACAGGCGAGTG 43 E207DAATATCGACTTTAGTCATCCAGATGTACAAGATGAGTTGAAGGA 44 F272SGACGTAGGTGCTCTCGAATCTTATTTAGATGAAATGAATTGGG 45 S375PCGATAACATTCCAGCTAAAAA 46 W434R GACCTGGTGGTTCCAAGAGAATGTATGTAGGACGTCAG 47E466D AATGGCGATGGATGGGGCGATTTCTTTACGAATGGAGGATCT 48 D128XCCAACGAATCGTTGGCAGNNNATTTCAGGTGCCTACACG 49 QEYKGTTGACTGGGATGAGCGCAAACAAGAAAATCAT 50 N190F TGGATGAAGAGTTCGGTAATTATGA 51Q209 AGTCATCCAGAGGTCGTAGATGAGTTGAAGGAT

[0071] By inserting an expression promoter region and the mutantα-amylase gene portion into the SmaI site of pKF19k again in a similarmanner as the above, the mutation-introduced gene became a templateplasmid upon introduction of another mutation. Another mutation was thusintroduced in a similar manner to the above-described method.

[0072] With these mutated recombinant plasmids thus obtained as atemplate, PCR reaction was conducted using primers CLUBG (SEQ ID No. 9)and LADH (SEQ ID No. 6) or primers CLUBS (SEQ ID No. 9) and K38DH (SEQID No. 8) to amplify the mutated gene fragments. After they were cutwith SalI, they were inserted into the site of SalI-SmaI site of anexpression vector pHSP64, whereby various plasmids for producing mutantα-amylases were constructed (FIG. 1).

Example 3

[0073] Production of Mutant α-Amylases

[0074] The various plasmids for producing mutant α-amylases obtained inExample 2 were each introduced into the strain Bacillus subtilis ISW1214(leuA metB5 hsdM1) in accordance with the protoplast method. Therecombinant Bacillus subtilis thus obtained was cultivated at 30° C. for4 days in a liquid medium (corn steep liquor, 4%; tryptose, 1%; meetextract, 1%, monopotassium phosphate, 0.1%, magnesium sulfate, 0.01%,maltose, 2%, calcium chloride, 0.1%, tetracycline, 15 μg/mL). Theactivity of each of the various mutant α-amylases was measured using thesupernatant of the culture medium.

Example 4

[0075] Evaluation of Amylase Productivity -1

[0076] Each of an enzyme having Pro₁₈ of ΔRG substituted with Ser (whichwill hereinafter be abbreviated as “P18S/ΔRG”), an enzyme having Gln₈₆substituted with Glu (“Q86E/ΔRG”), an enzyme having Glu₁₃₀ substitutedwith Val (“E130V/ΔRG”), an enzyme having Asn₁₅₄ substituted with Asp(“N154D/ΔRG”), an enzyme having Arg₁₇₁ substituted with Cys(“R171C/ΔRG”), an enzyme having Ala₁₈₆ substituted with Val(“A186V/ΔRG”), an enzyme having Glu₂₁₂ substituted with Asp(“E212D/ΔRG”), an enzyme having Val₂₂₂ substituted with Glu(“V222E/ΔRG”), an enzyme having Tyr₂₄₃ substituted with Cys(“Y243C/ΔRG”), an enzyme having Pro₂₆₀ substituted with Glu(“P260E/ΔRG”), an enzyme having Lys₂₆₉ substituted with Gln(“K269E/ΔRG”), an enzyme having Glu₂₇₆ substituted with His(“E276H/ΔRG”), an enzyme having Asn₂₇₇ substituted with Ser(“N277S/ΔRG”), an enzyme having Arg₃₁₀ substituted with Ala(“R310A/ΔRG”), an enzyme having Glu₃₆₀ substituted with Gln(“E360Q/ΔRG”), an enzyme having Gln₃₉₁ substituted with Glu(“Q391E/ΔRG”), an enzyme having Trp₄₃₉ substituted with Arg(“W439R/ΔRG”), an enzyme having Lys₄₄₄ substituted with Arg(“K444R/ΔRG”), an enzyme having Asn₄₇₁ substituted with Asp(“N471D/ΔRG”), and an enzyme having Gly₄₇₆ substituted with Asp(“G476D/ΔRG”) was assayed for amylase productivity. As a control, ΔRGwas employed. A relative value (%) of amylase productivity wasdetermined from the amylase productivity of ΔRG set at 100%. The resultsare shown in Table 3. TABLE 3 Relative amylase Enzyme productivity (%)ΔRG 100 P18S/ΔRG 277 Q86E/ΔRG 119 E130V/ΔRG 362 N154D/ΔRG 146 R171C/ΔRG235 A186V/ΔRG 485 E212D/ΔRG 327 V222E/ΔRG 135 Y243C/ΔRG 350 P260E/ΔRG142 K269Q/ΔRG 142 E276H/ΔRG 231 N277S/ΔRG 312 R310A/ΔRG 208 E360Q/ΔRG162 Q391E/ΔRG 127 W439R/ΔRG 312 K444R/ΔRG 112 N471D/ΔRG 292 G476D/ΔRG296

[0077] Any one of the mutant enzymes exhibited higher amylaseproductivity than ΔRG, indicating that mutation heightened productivityof α-amylase in recombinant Bacillus subtilis. In particular, theproductivity of each of E130V/ΔRG, A186V/ΔRG, E212D/ΔRG, Y243C/ΔRG,N277S/ΔRG and W439R/ΔRG was found to be at least 3 times greater thanthat of ΔRG and above all, A186V/ΔRG exhibited eminently highproductivity of almost 5 times greater than that of ΔRG.

Example 5

[0078] Evaluation of Amylase Productivity-2

[0079] In a similar manner to the methods described in Examples 1, 2 and3, each of an enzyme having Pro₁₈ of ΔRG/MT substituted with Thr (whichwill hereinafter be abbreviated as “P18T/ΔRG/MT”), an enzyme havingGln₈₆ substituted with Glu (“Q86E/ΔRG/MT”), an enzyme having Glu₁₃₀substituted with Val (“E130V/ΔRG/MT”), an enzyme having Ala₁₈₆substituted with Asn (“A186N/ΔRG/MT”), an enzyme having Tyr₂₄₃substituted with Ser (“Y243S/ΔRG/MT”), an enzyme having Asn₂₇₇substituted with Phe (“N277F/ΔRG/MT”), and an enzyme having Asn₄₇₁substituted with Glu (“N471E/ΔRG/MT”) was assayed for amylaseproductivity. As a control, ΔRG/MT was employed. The results are shownin Table 4. TABLE 4 Relative amylase Enzyme productivity (%) ΔRG/MT 100P18T/ΔRG/MT 200 Q86E/ΔRG/MT 144 E130V/ΔRG/MT 344 A186N/ΔRG/MT 344Y243S/ΔRG/MT 189 N277F/ΔRG/MT 256 N471E/ΔRG/MT 211

[0080] It was recognized that any one of the above-described mutantenzymes exhibited high amylase productivity compared with ΔRG/MT, and inparticular, the productivity of each of E130V/ΔRG/MT and A186N/ΔRG/MTwas at least 3 times greater than that of ΔRG/MT.

Example 6

[0081] Evaluation of Amylase Productivity-3

[0082] In accordance with the methods employed in Examples 1, 2 and 3,each of an enzyme having Asp₁₂₈ of K38AMY substituted with Val (whichwill hereinafter be abbreviated as “D128V”), an enzyme having Gly₁₄₀substituted with Ser (“G140S”), an enzyme having Ser₁₄₄ substituted withPro (“S144P”), an enzyme having Arg₁₆₈ substituted with Gln (“R168Q”),an enzyme having Asn₁₈₁ substituted with Val (“N181V”), an enzyme havingGlu₂₀₇ substituted with Asp (“E207D”), an enzyme having Phe₂₇₂substituted with Ser (“F272S”), an enzyme having Ser₃₇₅ substituted withPro (“S375P”), an enzyme having Trp₄₃₄ substituted with Arg (“W434R”),and an enzyme having Glu₄₆₆ substituted with Asp (“E466D”) was assayedfor amylase productivity. As a control, K38AMY was employed. The resultsare shown in Table 5. TABLE 5 Relative amylase Enzyme productivity (%)K38AMY 100 D128V 325 G140S 209 S144P 197 R168Q 264 N181V 207 E207D 109F272S 175 S375P 115 W434R 124 E466D 212

[0083] It was recognized that compared with the wild type K38AMY, anyone of the mutant enzymes exhibited high amylase productivity and inparticular, D128V exhibited high productivity at least 3 times greaterthan that of K38AMY.

Example 7

[0084] Evaluation of Amylase Productivity-4

[0085] A mutant enzyme S144P/N181V (which will hereinafter beabbreviated as “SPNV”) having, among the mutants shown in Example 6,S144P and N181V in combination was assayed for amylase productivity inaccordance with the method as described in Example 3. As a control,K38AMY, S144P and N181V were employed. The results are shown in Table 6.TABLE 6 Relative amylase Enzyme productivity (%) K38AMY 100 S144P 197N181V 207 SPNV 257

[0086] As a result, as shown in Table 6, a further improvement inamylase productivity was brought about by combined use.

Example 8

[0087] Evaluation of Amylase Productivity-5

[0088] In accordance with the methods as described in Examples 1, 2 and3, each of an enzyme obtained by substituting Arg₁₆₈ of the gene of aheat-resistance improved enzyme LA-K38AMY/NFQV with Gln (which willhereinafter be abbreviated as “R168Q/LA-K38AMY/NFQV”), an enzymeobtained by substituting Glu₄₆₆ of the above-described gene with Asp(“E466D/LA-K38AMY/NFQV”), and an enzyme having double mutations ofExample 6 introduced into the gene (“SPNV/LA-K38AMY/NFQV”) was assayedfor amylase productivity. As a control, LA-K38AMY/NFQV was employed. Theresults are shown in Table 7. TABLE 7 Relative amylase Enzymeproductivity (%) LA-K38AMY/NFQV 100 R168Q/LA-K38AMY/NFQV 304E466D/LA-K38AMY/NFQV 264 SPNV/LA-K38AMY/NFQV 154

[0089] As a result, it was recognized that any one of the mutant enzymesobtained in this Example exhibited high amylase productivity at leastabout 1.5 times greater than that of LA-K38AMY/NFQV and in particular,R168Q/LA-K38AMY/NFQV exhibited about 3 times greater productivity.

Example 9

[0090] Evaluation of Amylase Productivity-6

[0091] In accordance with the methods as described in Examples 1, 2 and3, each of an enzyme obtained by substituting Asp₁₂₈ of the gene of aheat-resistance improved enzyme LA-K38AMY/QEYK/NFQV with Val (which willhereinafter be abbreviated as “D128V/LA-K38AMY/QEYK/NFQV”) and an enzymehaving double mutations of Example 6 introduced into the gene(“SPNV/LA-K38AMY/QEYK/NFQV”) was assayed for amylase productivity. As acontrol, LA-K38AMY/QEYK/NFQV was employed. The results are shown inTable 8. TABLE 8 Relative amylase Enzyme productivity (%)LA-K38AMY/QEYK/NFQV 100 D128V/LA-K38AMY/QEYK/NFQV 602SPNV/LA-K38AMY/QEYK/NFQV 427

[0092] As a result, it was recognized that any one of the mutant enzymesobtained in this Example exhibited markedly high amylase productivitycompared with LA-K38AMY/QEYK/NFQV and in particular,D128V/LA-K38AMY/QEYK/NFQV exhibited drastic increase (about 6 times) inproductivity.

Example 10

[0093] Evaluation of Amylase Productivity-7

[0094] Into D128V/LA-K38AMY/QEYK/NFQV which was recognized to show adrastic increase in productivity among the mutant enzymes shown inExample 9, a mutation for heightening oxidant resistance by substitutingMet₁₀₇ with Leu (this mutation will hereinafter be abbreviated as“M107L”) was introduced in accordance with the methods as described inExamples 1 and 2 (“ML/DV/LA-K38AMY/QEYK/NFQV”).

[0095] Then, the gene of the mutant enzyme ML/DV/LA-K38AMY/QEYK/NFQV wasassayed for amylase productivity in accordance with the method ofExample 4. As a control, D128V/LA-K38AMY/QEYK/NFQV was employed. Theresults are shown in Table 9. TABLE 9 Relative amylase Enzymeproductivity (%) D128V/LA-K38AMY/QEYK/NFQV 100 M107L/D128V/LA- 115K38AMY/QEYK/NFQV

[0096] The relative amylase productivity of the mutant enzymeML/DV/LA-K38AMY/QEYK/NFQV was 115%, indicating that introduction ofM107L mutation for reinforcing oxidant resistance did not adverselyaffect high productivity of amylase in recombinant Bacillus subtilis.

Example 11

[0097] Evaluation of Amylase Productivity-8

[0098] In accordance with the methods as described in Examples 1, 2 and3, an enzyme obtained by substituting AsP₁₂₈ of the gene ofheat-resistance-improved enzyme QEYK/NFQV with Gln (the resultant enzymewill hereinafter be abbreviated as “D128Q/QEYK/NFQV”) was assayed foramylase productivity. As a control, QEYK/NFQV was employed. The resultsare shown in Table 10. TABLE 10 Relative amylase Enzyme productivity (%)QEYK/NFQV 100 D128Q/QEYK/NFQV 247

[0099] It was recognized that the mutant enzyme exhibited productivityof at least 2 times greater than that of QEYK/NFQV.

Example 12

[0100] Solubility Assay

[0101] After storage of each of the mutant enzyme preparations as shownin Table 11 at 4° C. for 1 week, the precipitate formed bycentrifugation (13000 rpm, 10 minutes, 4° C.) was separated. Theprecipitate was suspended in the same volume, as that beforecentrifugation, of a Tris-HCl buffer (pH 7.0) containing of 2 mM CaCl₂.The resulting suspension was diluted about 500-folds with the samebuffer to dissolve the former in the latter and enzymatic activity inthe resulting solution was measured. The supernatant was diluted in asimilar manner and enzymatic activity in it was also measured.Solubility of each of the mutant enzymes was evaluated by comparing theenzymatic activity in each of the precipitate solution and supernatantwith that of the preparation before storage at 4° C. The results areshown collectively in Table 11. TABLE 11 Residual activity (%) afterstorage at 4° C. Enzyme Supernatant Precipitate ΔRG 55 40 ΔRG Gln86 →Glu 83 11 ΔRG Pro260 → Glu 70 18 ΔRG Lys269 → Gln 74 27 ΔRG Asn471 → Asp74 23 ΔRG Lys484 → Gln 71 24

[0102] As a result, when an improved α-amylase (ΔRG) having heatresistance improved by deleting Arg₁₈₁ and Gly₁₈₂ was stored at 4° C.for one week, precipitation of the enzyme was recognized and only abouthalf of the activity remained in the supernatant. On the other hand, themutant enzymes obtained by introducing a further mutation in ΔRG-LAMYshowed a high activity residual ratio in the supernatant, indicating animprovement in solubility by mutation. In particular, the enzyme havingGln₈₆ substituted with Glu showed the highest enzyme solubility and 80%of the enzyme remained in the supernatant under the conditions of thisExample.

Example 13

[0103] Detergent Composition for Automatic Dish Washer

[0104] A detergent composition for automatic dish washer having thecomposition as shown in Table 12 was prepared, followed by incorporationtherein of various mutant enzymes obtained in the productivityincreasing method. As a result, the highly productive mutant enzymesexhibited similar or superior detergency to the control enzyme when theywere equal in activity. TABLE 12 Composition of detergent (%) PluronicL-61 2.2 Sodium carbonate 24.7 Sodium bicarbonate 24.7 Sodiumpercarbonate 10.0 No. 1 sodium silicate 12.0 Trisodium citrate 20.0Polypropylene glycol 2.2 “Silicone KST-04” (product of Toshiba Silicone)0.2 “Sokalan CP-45” (product of BASF) 4.0

[0105] Capability of Exploitation Industry

[0106] By using the mutant α-amylases according to the presentinvention, α-amylases are available at a high yield from recombinantmicroorganisms, making it possible to largely reduce the cost of theirindustrial production. The mutation for productivity increase in thepresent invention does not adversely affect biochemical properties ofthe enzymes so that highly productive liquefying alkaline α-amylaseshaving heat resistance, chelating agent resistance and oxidantresistance and being useful as enzymes for a detergent can be produced.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 51 <210> SEQ ID NO 1<211> LENGTH: 1786 <212> TYPE: DNA <213> ORGANISM: Bacillus sp.KSM-AP1378 <220> FEATURE: <221> NAME/KEY: sig_peptide <222> LOCATION:(155)..(247) <223> OTHER INFORMATION: <221> NAME/KEY: mat_peptide <222>LOCATION: (248)..() <223> OTHER INFORMATION: <221> NAME/KEY: CDS <222>LOCATION: (155)..(1702) <223> OTHER INFORMATION: <400> SEQUENCE: 1cagcgtgata atataaattt gaaatgaaca cctatgaaaa tatggtagcg attgcgcgac 60gagaaaaaac ttgggagtta ggaagtgata ttaaaggatt ttttttgact tgttgtgaaa 120acgcttgcat aaattgaagg agagggtgct tttt atg aaa ctt cat aac cgt ata 175Met Lys Leu His Asn Arg Ile -30 -25 att agc gta cta tta aca cta ttg ttagct gta gct gtt ttg ttt cca 223 Ile Ser Val Leu Leu Thr Leu Leu Leu AlaVal Ala Val Leu Phe Pro -20 -15 -10 tat atg acg gaa cca gca caa gcc catcat aat ggg acg aat ggg acc 271 Tyr Met Thr Glu Pro Ala Gln Ala His HisAsn Gly Thr Asn Gly Thr -5 -1 1 5 atg atg cag tat ttt gaa tgg cat ttgcca aat gac ggg aac cac tgg 319 Met Met Gln Tyr Phe Glu Trp His Leu ProAsn Asp Gly Asn His Trp 10 15 20 aac agg tta cga gat gac gca gct aac ttaaag agt aaa ggg att acc 367 Asn Arg Leu Arg Asp Asp Ala Ala Asn Leu LysSer Lys Gly Ile Thr 25 30 35 40 gct gtt tgg att cct cct gca tgg aag gggact tcg caa aat gat gtt 415 Ala Val Trp Ile Pro Pro Ala Trp Lys Gly ThrSer Gln Asn Asp Val 45 50 55 ggg tat ggt gcc tat gat ttg tac gat ctt ggtgag ttt aac caa aag 463 Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly GluPhe Asn Gln Lys 60 65 70 gga acc gtc cgt aca aaa tat ggc aca agg agt cagttg caa ggt gcc 511 Gly Thr Val Arg Thr Lys Tyr Gly Thr Arg Ser Gln LeuGln Gly Ala 75 80 85 gtg aca tct ttg aaa aat aac ggg att caa gtt tat ggggat gtc gtg 559 Val Thr Ser Leu Lys Asn Asn Gly Ile Gln Val Tyr Gly AspVal Val 90 95 100 atg aat cat aaa ggt gga gca gac ggg aca gag atg gtaaat gcg gtg 607 Met Asn His Lys Gly Gly Ala Asp Gly Thr Glu Met Val AsnAla Val 105 110 115 120 gaa gtg aac cga agc aac cga aac caa gaa ata tcaggt gaa tac acc 655 Glu Val Asn Arg Ser Asn Arg Asn Gln Glu Ile Ser GlyGlu Tyr Thr 125 130 135 att gaa gca tgg acg aaa ttt gat ttc cct gga agagga aat acc cat 703 Ile Glu Ala Trp Thr Lys Phe Asp Phe Pro Gly Arg GlyAsn Thr His 140 145 150 tcc aac ttt aaa tgg cgc tgg tat cat ttt gat gggaca gat tgg gat 751 Ser Asn Phe Lys Trp Arg Trp Tyr His Phe Asp Gly ThrAsp Trp Asp 155 160 165 cag tca cgt cag ctt cag aac aaa ata tat aaa ttcaga ggt acc gga 799 Gln Ser Arg Gln Leu Gln Asn Lys Ile Tyr Lys Phe ArgGly Thr Gly 170 175 180 aag gca tgg gac tgg gaa gta gat ata gag aac ggcaac tat gat tac 847 Lys Ala Trp Asp Trp Glu Val Asp Ile Glu Asn Gly AsnTyr Asp Tyr 185 190 195 200 ctt atg tat gca gac att gat atg gat cat ccagaa gta atc aat gaa 895 Leu Met Tyr Ala Asp Ile Asp Met Asp His Pro GluVal Ile Asn Glu 205 210 215 ctt aga aat tgg gga gtt tgg tat aca aat acactt aat cta gat gga 943 Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn Thr LeuAsn Leu Asp Gly 220 225 230 ttt aga atc gat gct gtg aaa cat att aaa tacagc tat acg aga gat 991 Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr SerTyr Thr Arg Asp 235 240 245 tgg cta aca cat gtg cgt aac acc aca ggt aaacca atg ttt gca gtt 1039 Trp Leu Thr His Val Arg Asn Thr Thr Gly Lys ProMet Phe Ala Val 250 255 260 gca gaa ttt tgg aaa aat gac ctt gct gca atcgaa aac tat tta aat 1087 Ala Glu Phe Trp Lys Asn Asp Leu Ala Ala Ile GluAsn Tyr Leu Asn 265 270 275 280 aaa aca agt tgg aat cac tcc gtg ttc gatgtt cct ctt cat tat aat 1135 Lys Thr Ser Trp Asn His Ser Val Phe Asp ValPro Leu His Tyr Asn 285 290 295 ttg tac aat gca tct aat agt ggt ggc tatttt gat atg aga aat att 1183 Leu Tyr Asn Ala Ser Asn Ser Gly Gly Tyr PheAsp Met Arg Asn Ile 300 305 310 tta aat ggt tct gtc gta caa aaa cac cctata cat gca gtc aca ttt 1231 Leu Asn Gly Ser Val Val Gln Lys His Pro IleHis Ala Val Thr Phe 315 320 325 gtt gat aac cat gac tct cag cca gga gaagca ttg gaa tcc ttt gtt 1279 Val Asp Asn His Asp Ser Gln Pro Gly Glu AlaLeu Glu Ser Phe Val 330 335 340 caa tcg tgg ttc aaa cca ctg gca tat gcattg att ctg aca agg gag 1327 Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala LeuIle Leu Thr Arg Glu 345 350 355 360 caa ggt tac cct tcc gta ttt tac ggtgat tac tac ggt ata cca act 1375 Gln Gly Tyr Pro Ser Val Phe Tyr Gly AspTyr Tyr Gly Ile Pro Thr 365 370 375 cat ggt gtt cct tcg atg aaa tct aaaatt gat cca ctt ctg cag gca 1423 His Gly Val Pro Ser Met Lys Ser Lys IleAsp Pro Leu Leu Gln Ala 380 385 390 cgt caa acg tat gcc tac gga acc caacat gat tat ttt gat cat cat 1471 Arg Gln Thr Tyr Ala Tyr Gly Thr Gln HisAsp Tyr Phe Asp His His 395 400 405 gat att atc ggc tgg acg aga gaa ggggac agc tcc cac cca aat tca 1519 Asp Ile Ile Gly Trp Thr Arg Glu Gly AspSer Ser His Pro Asn Ser 410 415 420 gga ctt gca act att atg tcc gat gggcca ggg ggt aat aaa tgg atg 1567 Gly Leu Ala Thr Ile Met Ser Asp Gly ProGly Gly Asn Lys Trp Met 425 430 435 440 tat gtc ggg aaa cat aaa gct ggccaa gta tgg aga gat atc acc gga 1615 Tyr Val Gly Lys His Lys Ala Gly GlnVal Trp Arg Asp Ile Thr Gly 445 450 455 aat agg tct ggt acc gtc acc attaat gca gat ggt tgg ggg aat ttc 1663 Asn Arg Ser Gly Thr Val Thr Ile AsnAla Asp Gly Trp Gly Asn Phe 460 465 470 act gta aac gga ggg gca gtt tcggtt tgg gtg aag caa taaataagga 1712 Thr Val Asn Gly Gly Ala Val Ser ValTrp Val Lys Gln 475 480 485 acaagaggcg aaaattactt tcctacatgc agagctttccgatcactcat acacccaata 1772 taaattggaa gctt 1786 <210> SEQ ID NO 2 <211>LENGTH: 516 <212> TYPE: PRT <213> ORGANISM: Bacillus sp. KSM-AP1378<400> SEQUENCE: 2 Met Lys Leu His Asn Arg Ile Ile Ser Val Leu Leu ThrLeu Leu Leu -30 -25 -20 Ala Val Ala Val Leu Phe Pro Tyr Met Thr Glu ProAla Gln Ala His -15 -10 -5 -1 1 His Asn Gly Thr Asn Gly Thr Met Met GlnTyr Phe Glu Trp His Leu 5 10 15 Pro Asn Asp Gly Asn His Trp Asn Arg LeuArg Asp Asp Ala Ala Asn 20 25 30 Leu Lys Ser Lys Gly Ile Thr Ala Val TrpIle Pro Pro Ala Trp Lys 35 40 45 Gly Thr Ser Gln Asn Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr Asp 50 55 60 65 Leu Gly Glu Phe Asn Gln Lys Gly ThrVal Arg Thr Lys Tyr Gly Thr 70 75 80 Arg Ser Gln Leu Gln Gly Ala Val ThrSer Leu Lys Asn Asn Gly Ile 85 90 95 Gln Val Tyr Gly Asp Val Val Met AsnHis Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Met Val Asn Ala Val GluVal Asn Arg Ser Asn Arg Asn Gln 115 120 125 Glu Ile Ser Gly Glu Tyr ThrIle Glu Ala Trp Thr Lys Phe Asp Phe 130 135 140 145 Pro Gly Arg Gly AsnThr His Ser Asn Phe Lys Trp Arg Trp Tyr His 150 155 160 Phe Asp Gly ThrAsp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys Ile 165 170 175 Tyr Lys PheArg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Ile 180 185 190 Glu AsnGly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp 195 200 205 HisPro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr 210 215 220225 Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile 230235 240 Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr245 250 255 Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp LeuAla 260 265 270 Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His SerVal Phe 275 280 285 Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser AsnSer Gly Gly 290 295 300 305 Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly SerVal Val Gln Lys His 310 315 320 Pro Ile His Ala Val Thr Phe Val Asp AsnHis Asp Ser Gln Pro Gly 325 330 335 Glu Ala Leu Glu Ser Phe Val Gln SerTrp Phe Lys Pro Leu Ala Tyr 340 345 350 Ala Leu Ile Leu Thr Arg Glu GlnGly Tyr Pro Ser Val Phe Tyr Gly 355 360 365 Asp Tyr Tyr Gly Ile Pro ThrHis Gly Val Pro Ser Met Lys Ser Lys 370 375 380 385 Ile Asp Pro Leu LeuGln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr Gln 390 395 400 His Asp Tyr PheAsp His His Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415 Asp Ser SerHis Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly 420 425 430 Pro GlyGly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly Gln 435 440 445 ValTrp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn 450 455 460465 Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser Val 470475 480 Trp Val Lys Gln 485 <210> SEQ ID NO 3 <211> LENGTH: 1753 <212>TYPE: DNA <213> ORGANISM: Bacillus sp. KSM-K38 <220> FEATURE: <221>NAME/KEY: sig_peptide <222> LOCATION: (162)..(224) <223> OTHERINFORMATION: <221> NAME/KEY: mat_peptide <222> LOCATION: (225)..() <223>OTHER INFORMATION: <221> NAME/KEY: CDS <222> LOCATION: (162)..(1664)<223> OTHER INFORMATION: <400> SEQUENCE: 3 gtatgcgaaa cgatgcgcaaaactgcgcaa ctactagcac tcttcaggga ctaaaccacc 60 ttttttccaa aaatgacatcatataaacaa atttgtctac caatcactat ttaaagctgt 120 ttatgatata tgtaagcgttatcattaaaa ggaggtattt g atg aga aga tgg gta 176 Met Arg Arg Trp Val -20gta gca atg ttg gca gtg tta ttt tta ttt cct tcg gta gta gtt gca 224 ValAla Met Leu Ala Val Leu Phe Leu Phe Pro Ser Val Val Val Ala -15 -10 -5-1 gat gga ttg aac ggt acg atg atg cag tat tat gag tgg cat ttg gaa 272Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu 1 5 1015 aac gac ggg cag cat tgg aat cgg ttg cac gat gat gcc gca gct ttg 320Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu 20 25 30agt gat gct ggt att aca gct att tgg att ccg cca gcc tac aaa ggt 368 SerAsp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 aatagt cag gcg gat gtt ggg tac ggt gca tac gat ctt tat gat tta 416 Asn SerGln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 gga gagttc aat caa aag ggt act gtt cga acg aaa tac gga act aag 464 Gly Glu PheAsn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 gca cagctt gaa cga gct att ggg tcc ctt aaa tct aat gat atc aat 512 Ala Gln LeuGlu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90 95 gta tac ggagat gtc gtg atg aat cat aaa atg gga gct gat ttt acg 560 Val Tyr Gly AspVal Val Met Asn His Lys Met Gly Ala Asp Phe Thr 100 105 110 gag gca gtgcaa gct gtt caa gta aat cca acg aat cgt tgg cag gat 608 Glu Ala Val GlnAla Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp 115 120 125 att tca ggtgcc tac acg att gat gcg tgg acg ggt ttc gac ttt tca 656 Ile Ser Gly AlaTyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser 130 135 140 ggg cgt aacaac gcc tat tca gat ttt aag tgg aga tgg ttc cat ttt 704 Gly Arg Asn AsnAla Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe 145 150 155 160 aat ggtgtt gac tgg gat cag cgc tat caa gaa aat cat att ttc cgc 752 Asn Gly ValAsp Trp Asp Gln Arg Tyr Gln Glu Asn His Ile Phe Arg 165 170 175 ttt gcaaat acg aac tgg aac tgg cga gtg gat gaa gag aac ggt aat 800 Phe Ala AsnThr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190 tat gattac ctg tta gga tcg aat atc gac ttt agt cat cca gaa gta 848 Tyr Asp TyrLeu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205 caa gatgag ttg aag gat tgg ggt agc tgg ttt acc gat gag tta gat 896 Gln Asp GluLeu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215 220 ttg gatggt tat cgt tta gat gct att aaa cat att cca ttc tgg tat 944 Leu Asp GlyTyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr 225 230 235 240 acatct gat tgg gtt cgg cat cag cgc aac gaa gca gat caa gat tta 992 Thr SerAsp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu 245 250 255 tttgtc gta ggg gaa tat tgg aag gat gac gta ggt gct ctc gaa ttt 1040 Phe ValVal Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe 260 265 270 tattta gat gaa atg aat tgg gag atg tct cta ttc gat gtt cca ctt 1088 Tyr LeuAsp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu 275 280 285 aattat aat ttt tac cgg gct tca caa caa ggt gga agc tat gat atg 1136 Asn TyrAsn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met 290 295 300 cgtaat att tta cga gga tct tta gta gaa gcg cat ccg atg cat gca 1184 Arg AsnIle Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala 305 310 315 320gtt acg ttt gtt gat aat cat gat act cag cca ggg gag tca tta gag 1232 ValThr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu 325 330 335tca tgg gtt gct gat tgg ttt aag cca ctt gct tat gcg aca att ttg 1280 SerTrp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu 340 345 350acg cgt gaa ggt ggt tat cca aat gta ttt tac ggt gat tac tat ggg 1328 ThrArg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly 355 360 365att cct aac gat aac att tca gct aaa aaa gat atg att gat gag ctg 1376 IlePro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu 370 375 380ctt gat gca cgt caa aat tac gca tat ggc acg cag cat gac tat ttt 1424 LeuAsp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe 385 390 395400 gat cat tgg gat gtt gta gga tgg act agg gaa gga tct tcc tcc aga 1472Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg 405 410415 cct aat tca ggc ctt gcg act att atg tcg aat gga cct ggt ggt tcc 1520Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser 420 425430 aag tgg atg tat gta gga cgt cag aat gca gga caa aca tgg aca gat 1568Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp 435 440445 tta act ggt aat aac gga gcg tcc gtt aca att aat ggc gat gga tgg 1616Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp 450 455460 ggc gaa ttc ttt acg aat gga gga tct gta tcc gtg tac gtg aac caa 1664Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln 465 470475 480 taacaaaaag ccttgagaag ggattcctcc ctaactcaag gctttctttatgtcgcttag 1724 cttaacgctt ctacgacttt gaagcttta 1753 <210> SEQ ID NO 4<211> LENGTH: 501 <212> TYPE: PRT <213> ORGANISM: Bacillus sp. KSM-K38<400> SEQUENCE: 4 Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu PheLeu Phe Pro -20 -15 -10 Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr MetMet Gln Tyr Tyr -5 -1 1 5 10 Glu Trp His Leu Glu Asn Asp Gly Gln His TrpAsn Arg Leu His Asp 15 20 25 Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile ThrAla Ile Trp Ile Pro 30 35 40 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp ValGly Tyr Gly Ala Tyr 45 50 55 Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln LysGly Thr Val Arg Thr 60 65 70 75 Lys Tyr Gly Thr Lys Ala Gln Leu Glu ArgAla Ile Gly Ser Leu Lys 80 85 90 Ser Asn Asp Ile Asn Val Tyr Gly Asp ValVal Met Asn His Lys Met 95 100 105 Gly Ala Asp Phe Thr Glu Ala Val GlnAla Val Gln Val Asn Pro Thr 110 115 120 Asn Arg Trp Gln Asp Ile Ser GlyAla Tyr Thr Ile Asp Ala Trp Thr 125 130 135 Gly Phe Asp Phe Ser Gly ArgAsn Asn Ala Tyr Ser Asp Phe Lys Trp 140 145 150 155 Arg Trp Phe His PheAsn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 160 165 170 Asn His Ile PheArg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 175 180 185 Glu Glu AsnGly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 190 195 200 Ser HisPro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe 205 210 215 ThrAsp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 220 225 230235 Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 240245 250 Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val255 260 265 Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met SerLeu 270 275 280 Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser GlnGln Gly 285 290 295 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser LeuVal Glu Ala 300 305 310 315 His Pro Met His Ala Val Thr Phe Val Asp AsnHis Asp Thr Gln Pro 320 325 330 Gly Glu Ser Leu Glu Ser Trp Val Ala AspTrp Phe Lys Pro Leu Ala 335 340 345 Tyr Ala Thr Ile Leu Thr Arg Glu GlyGly Tyr Pro Asn Val Phe Tyr 350 355 360 Gly Asp Tyr Tyr Gly Ile Pro AsnAsp Asn Ile Ser Ala Lys Lys Asp 365 370 375 Met Ile Asp Glu Leu Leu AspAla Arg Gln Asn Tyr Ala Tyr Gly Thr 380 385 390 395 Gln His Asp Tyr PheAsp His Trp Asp Val Val Gly Trp Thr Arg Glu 400 405 410 Gly Ser Ser SerArg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 415 420 425 Gly Pro GlyGly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly 430 435 440 Gln ThrTrp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile 445 450 455 AsnGly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 460 465 470475 Val Tyr Val Asn Gln 480 <210> SEQ ID NO 5 <211> LENGTH: 30 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 5 gagtcgacca gcacaagcccatcataatgg 30 <210> SEQ ID NO 6 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic DNA <400> SEQUENCE: 6 taaagcttca atttatattg g 21 <210> SEQ IDNO 7 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400>SEQUENCE: 7 gggtcgacca gcacaagccg atggattgaa cggtacgatg 40 <210> SEQ IDNO 8 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400>SEQUENCE: 8 taaagctttt gttattggtt cacgtacac 29 <210> SEQ ID NO 9 <211>LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 9ccagatctac ttaccatttt agagtca 27 <210> SEQ ID NO 10 <211> LENGTH: 34<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 10 atttgccaaatgacgggcag cattggaatc ggtt 34 <210> SEQ ID NO 11 <211> LENGTH: 34 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 11 aaccgattcc aatgctgcccgtcatttggc aaat 34 <210> SEQ ID NO 12 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 12 tttgaatggc atttgtcaaatgacggggaa ccac 34 <210> SEQ ID NO 13 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 13 acaaggagtc agttggaaggtgccgtgaca tct 33 <210> SEQ ID NO 14 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 14 cgaaaccaag taatatcagg t 21<210> SEQ ID NO 15 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 15 aatacccatt ccgattttaa atggcgc 27 <210> SEQ ID NO16 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 16gattgggatc agtcatgyca gcttcagaac aaa 33 <210> SEQ ID NO 17 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 17 aaattcaccggaaaggtatg ggactgggaa gta 33 <210> SEQ ID NO 18 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 18 tcatccagat gtaatcaatg 20<210> SEQ ID NO 19 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 19 cttagaaatt ggggagaatg gtatacaaat aca 33 <210> SEQID NO 20 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400>SEQUENCE: 20 gtgaaacata ttaaatgcag ctatacgaga gat 33 <210> SEQ ID NO 21<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 21aacaccacag gtaaagaaat gtttgcagtt gca 33 <210> SEQ ID NO 22 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 22 agaattttggcaaaatgacc t 21 <210> SEQ ID NO 23 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 23 ttgctgcaat ccataactatttaaat 26 <210> SEQ ID NO 24 <211> LENGTH: 33 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic DNA <400> SEQUENCE: 24 cttgctgcaa tcgaaagyta tttaaataaa aca 33<210> SEQ ID NO 25 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 25 ggctattttg atatggcaaa tattttaaat ggt 33 <210> SEQID NO 26 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400>SEQUENCE: 26 tctgacaagg cagcaaggtt a 21 <210> SEQ ID NO 27 <211> LENGTH:27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 27 gatccacttctggaagcacg tcaaacg 27 <210> SEQ ID NO 28 <211> LENGTH: 27 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 28 gggggtaata aaagaatgtatgtcggg 27 <210> SEQ ID NO 29 <211> LENGTH: 26 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic DNA <400> SEQUENCE: 29 atgtatgtcg ggcgacataa agctgg 26 <210>SEQ ID NO 30 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400>SEQUENCE: 30 gatggttggg gggatttcac tgtaa 25 <210> SEQ ID NO 31 <211>LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 31ttcactgtaa acgatggggc agtttcg 27 <210> SEQ ID NO 32 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 32 ggtttgggtgcagcaataaa t 21 <210> SEQ ID NO 33 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <221> NAME/KEY: misc_feature <222> LOCATION:(16)..(18) <223> OTHER INFORMATION: n = a, c, t, or g <400> SEQUENCE: 33tttgaatggc atttgnnnaa tgacgggaac cac 33 <210> SEQ ID NO 34 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic DNA NAME/KEY: misc_feature <222>LOCATION: (16)..(18) <223> OTHER INFORMATION: n = a, c, t, or g <400>SEQUENCE: 34 aaattcaccg gaaagnnntg ggactgggaa gta 33 <210> SEQ ID NO 35<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <221> NAME/KEY:misc_feature <222> LOCATION: (16)..(18) <223> OTHER INFORMATION: n = a,c, t, or g <400> SEQUENCE: 35 gtgaaacata ttaaannnag ctatacgaga gat 33<210> SEQ ID NO 36 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <221> NAME/KEY: misc_feature <222> LOCATION: (16)..(18) <223> OTHERINFORMATION: n = a, c, t, or g <400> SEQUENCE: 36 cttgctgcaa tcgaannntatttaaataaa aca 33 <210> SEQ ID NO 37 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic DNA <400> SEQUENCE: 37 gatggttggg gggaattcactgtaa 25 SEQ ID NO 38 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 38 ccaacgaatc gttggcaggt aatttcaggt gcctacacg 39<210> SEQ ID NO 39 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 39 attgatgcgt ggacgagttt cgacttttca ggg 33 <210> SEQID NO 40 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400>SEQUENCE: 40 tttcgacttt ccagggcgta a 21 <210> SEQ ID NO 41 <211> LENGTH:43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 41 ggtgttgactgggatcagca atatcaagaa aatcatattt tcc 43 <210> SEQ ID NO 42 <211> LENGTH:42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic DNA <221> NAME/KEY: misc_feature<222> LOCATION: (27)..(27) <223> OTHER INFORMATION: n = a, c, t, or g<400> SEQUENCE: 42 catattttcc gctttgcaaa tacggtntgg aacaggcgag tg 42<210> SEQ ID NO 43 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 43 aatatcgact ttagtcatcc agatgtacaa gatgagttga agga44 <210> SEQ ID NO 44 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 44 gacgtaggtg ctctcgaatc ttatttagat gaaatgaatt ggg43 <210> SEQ ID NO 45 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 45 cgataacatt ccagctaaaa a 21 <210> SEQ ID NO 46<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 46gacctggtgg ttccaagaga atgtatgtag gacgtcag 38 <210> SEQ ID NO 47 <211>LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 47aatggcgatg gatggggcga tttctttacg aatggaggat ct 42 <210> SEQ ID NO 48<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <221> NAME/KEY:misc_feature <222> LOCATION: (19)..(21) <223> OTHER INFORMATION: n = a,c, t, or g <400> SEQUENCE: 48 ccaacgaatc gttggcagnn natttcaggt gcctacacg39 <210> SEQ ID NO 49 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SyntheticDNA <400> SEQUENCE: 49 gttgactggg atgagcgcaa acaagaaaat cat 33 <210> SEQID NO 50 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400>SEQUENCE: 50 tggatgaaga gttcggtaat tatga 25 <210> SEQ ID NO 51 <211>LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 51agtcatccag aggtcgtaga tgagttgaag gat 33

1. A mutant α-amylase which is derived from an α-amylase having an aminoacid sequence represented by SEQ ID No. 1 or showing at least 60%homology thereto by substitution or deletion of at least one amino acidresidue corresponding to any one of Pro₁₈, Gln₈₆, Glu₁₃₀, Asn₁₅₄,Arg₁₇₁, Ala186, Glu₂₁₂, Val₂₂₂, Tyr₂₄₃, Pro₂₆₀, Lys₂₆₉, GlU₂₇₆, Asn₂₇₇,Arg₃₁₀, Glu₃₆₀, Gln₃₉₁, Trp₄₃₉, Lys₄₄₄, Asn₄₇₁, and Gly₄₇₆ of the aminoacid sequence.
 2. A mutant α-amylase derived from an α-amylase having anamino acid sequence represented by SEQ ID No. 2 or showing at least 60%homology thereto by substitution or deletion of at least one amino acidresidue corresponding to any one of Asp₁₂₈, Gly₁₄₀, Ser₁₄₄, Arg₁₆₈,Asn₁₈₁, Glu₂₀₇, Phe₂₇₂, Ser₃₇₅, Trp₄₃₄ and Glu₄₆₆ of the amino acidsequence.
 3. A mutant α-amylase according to claim 1, wherein thesubstitution or deletion of at least one amino acid residue issubstitution of the amino acid residue corresponding to Pro₁₈ with Seror Thr, the amino acid residue corresponding to Gln₈₆ with Glu, theamino acid residue corresponding to Glu₁₃₀ with Val or Gln, the aminoacid residue corresponding to Asn₁₅₄ with Asp, the amino acid residuecorresponding to Arg₁₇₁ with Cys or Gln, the amino acid residuecorresponding to Ala₁₈₆ with Val or Asn, the amino acid residuecorresponding to Glu₂₁₂ with Asp, the amino acid residue correspondingto Val₂₂₂ with Glu, the amino acid residue corresponding to Tyr₂₄₃ withCys or Ser, the amino acid residue corresponding to Pro₂₆₀ with Glu, theamino acid residue corresponding to Lys₂₆₉ with Gln, the amino acidresidue corresponding to Glu₂₇₆ with His, the amino acid residuecorresponding to Asn₂₇₇ with Ser or Phe, the amino acid residuecorresponding to Arg₃₁₀ with Ala, the amino acid residue correspondingto Glu₃₆₀ with Gln, the amino acid residue corresponding to Gln₃₉₁ withGlu, the amino acid residue corresponding to Trp₄₃₉ with Arg, the aminoacid residue corresponding to Lys₄₄₄ with Arg, the amino acid residuecorresponding to Asn₄₇₁ with Asp or Glu, or the amino acid residuecorresponding to Gly₄₇₆ with Asp.
 4. A mutant α-amylase according toclaim 2, wherein the substitution or deletion of at least one amino acidresidue is substitution of the amino acid residue corresponding toAsp₁₂₈ with Val or Gln, the amino acid residue corresponding to Gly₁₄₀with Ser, the amino acid residue corresponding to Ser₁₄₄ with Pro, theamino acid residue corresponding to Arg₁₆₈ with Gln, the amino acidresidue corresponding to Gln₁₈₁ with Val, the amino acid residuecorresponding to Glu₂₇₀ with Asp, the amino acid residue correspondingto Phe₂₇₂ with Ser, the amino acid residue corresponding to Ser₃₇₅ withPro, the amino acid residue corresponding to Trp₄₃₄ with Arg or theamino acid residue corresponding to Glu₄₆₆ with Asp.
 5. A gene encodinga mutant α-amylase as claimed in any one of claims 1 to 4, or a vectorcontaining said gene.
 6. A cell transformed by a vector as claimed inclaim
 5. 7. A method for producing a mutant α-amylase, which comprisescultivating a transformant cell as claimed in claim
 6. 8. A detergentcomposition comprising a mutant α-amylase as claimed in any one ofclaims 1 to 4.